Methods for enhancing the specific uptake of botulinum neurotoxins into cells

Abstract

The present invention provides a method for enhancing the specific uptake of a neurotoxin polypeptide into cells, the method comprising: incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, the incubation comprising at least one of the following steps: (i) K.sup.+-mediated depolarization of the cells, (ii) a reduced neurotoxin polypeptide exposition time and/or (iii) agitation of the cells during neurotoxin polypeptide exposition, thereby enhancing the specific uptake of the neurotoxin polypeptide into said cells. In addition, the invention pertains to a method for directly determining the biological activity of a neurotoxin polypeptide in cells, comprising: a) incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, the incubation comprising at least one of the following steps: (i) K.sup.+-mediated depolarization of the cells, (ii) a reduced neurotoxin polypeptide exposition time and/or (iii) agitation of the cells during neurotoxin polypeptide exposition; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes and with at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d); and f) calculating the amount of substrate cleaved by said neurotoxin polypeptide in said cells by means of the second detection complexes, thereby determining the biological activity of said neurotoxin polypeptide in said cells.

Claims

1. A method for enhancing the specific uptake of a neurotoxin polypeptide into cells, the method comprising: incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, the incubation comprising at least two of the following steps: (i) K.sup.+-mediated depolarization of the cells, (ii) a reduced neurotoxin polypeptide exposition time and/or (iii) agitation of the cells during neurotoxin polypeptide exposition, thereby enhancing the specific uptake of the neurotoxin polypeptide into said cells.

2. The method of claim 1, followed by determining the biological activity of the neurotoxin polypeptide in the cells.

3. A method for directly determining the biological activity of a neurotoxin polypeptide in cells, comprising: a) incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, the incubation comprising at least two of the following steps: (i) K.sup.+-mediated depolarization of the cells, (ii) a reduced neurotoxin polypeptide exposition time and/or (iii) agitation of the cells during neurotoxin polypeptide exposition; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to a non-cleaved and neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes and with at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d); and f) calculating the amount of substrate cleaved by said neurotoxin polypeptide in said cells by means of the second detection complexes, thereby determining the biological activity of said neurotoxin polypeptide in said cells.

4. The method of claim 3, wherein the K.sup.+-mediated depolarization of the cells is carried out at an additional K.sup.+ concentration of about 20 mM to about 55 mM, for at least 2 hours.

5. The method of claim 3, wherein the K.sup.+-mediated depolarization of the cells and/or the neurotoxin polypeptide exposition is carried out in the presence of GT1b.

6. The method of claim 5, wherein GT1b is used in a concentration between 15 and 50 M.

7. The method of claim 3, wherein the reduced neurotoxin polypeptide exposition time is exposition of the cells to the neurotoxin polypeptide for at least 24 hours and less than 96 hours.

8. The method of claim 3, wherein agitation of the cells during neurotoxin polypeptide exposition is carried out with a magnetic stirrer, rotating spinner flasks or shaking of the cells by a shaker.

9. The method of claim 3, wherein the K.sup.+-mediated depolarization of the cells is carried out at an additional K.sup.+ concentration of about 20 mM to about 55 mM, for at least 2 hours in the presence of 20 M GT1b and neurotoxin polypeptide, followed by neurotoxin polypeptide exposition for additional 70 hours under agitation.

10. The method of claim 3, wherein an incubation time without neurotoxin polypeptide precedes the neurotoxin polypeptide exposition.

11. The method of claim 10, wherein the incubation time without neurotoxin polypeptide is between 16 and 48 hours.

12. The method of claim 3, wherein the method is a fluorescence method.

13. The method of claim 3, wherein the neurotoxin polypeptide is BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H, or subtypes thereof.

14. The method of claim 3, wherein the substrate is VAMP/Synaptobrevin, SNAP-25 or Syntaxin.

15. The method of claim 3, wherein the cells are neuronal cells or neuronal differentiated cells selected from the group consisting of: primary neuronal cells, tumor cells which are capable of differentiating to neuronal cells, P19 cells or induced pluripotent stem cell (IPS)-derived neurons.

16. The method of claim 1, wherein the K.sup.+-mediated depolarization of the cells is carried out at an additional K.sup.+ concentration of about 20 mM to about 55 mM, for at least 2 hours.

17. The method of claim 1, wherein the K.sup.+-mediated depolarization of the cells and/or the neurotoxin polypeptide exposition is carried out in the presence of GT1b.

18. The method of claim 1, wherein the reduced neurotoxin polypeptide exposition time is exposition of the cells to the neurotoxin polypeptide for at least 24 hours and less than 96 hours.

19. The method of claim 1, wherein agitation of the cells during neurotoxin polypeptide exposition is carried out with a magnetic stirrer, rotating spinner flasks or shaking of the cells by a shaker.

20. The method of claim 1, wherein the K.sup.+-mediated depolarization of the cells is carried out at an additional K.sup.+ concentration of at least about 20 mM to about 55 mM, for at least-2 hours in the presence of 20 M GT1b and neurotoxin polypeptide, followed by neurotoxin polypeptide exposition for additional 70 hours under agitation.

21. The method of claim 1, wherein an incubation time without neurotoxin polypeptide precedes the neurotoxin polypeptide exposition.

22. The method of claim 1, wherein the neurotoxin polypeptide is BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H, or subtypes thereof.

23. The method of claim 1, wherein the cells are neuronal cells or neuronal differentiated cells selected from the group consisting of: primary neuronal cells, tumor cells which are capable of differentiating to neuronal cells, P19 cells or induced pluripotent stem cell (IPS)-derived neurons.

24. The method of claim 15, wherein the tumor cells which are capable of differentiating to neuronal cells are neuroblastoma cells.

25. The method of claim 23, wherein the tumor cells which are capable of differentiating to neuronal cells are neuroblastoma cells.

Description

(1) The FIGURE shows:

(2) FIG. 1: Kinetics in loss of activity of stressed drug product samples. Drug product samples containing BoNT/A were stored at 70 C. for up to four weeks. After 0, 1, 2 and 4 weeks samples were drawn and subjected to analysis in the mouse LD.sub.50 bioassay as well as in the cell based assay (CBA) employing different protocols. On the x-axis the storage time in weeks is given whereas on the y-axis the relative potency is given. The potency at the start point was set to 100% and the consecutive test time points are expressed relative to the start point. The values for the LD.sub.50 bioassay are depicted as diamonds. The cell based assays protocol employing K.sup.+-depolarization is depicted in squares, the protocol employing an 8 hour toxin incubation time followed by a 64 hour toxin-free incubation time is depicted in triangles and the protocol employing shaking during the 72 hour incubation time is depicted in circles. A CBA protocol which was not modified is depicted in line symbols. In sum, FIG. 1 shows a comparison of three cell based assay examples where the stressed samples show a comparable kinetic in decay to the referenced assay, the mouse LD.sub.50 bioassay.

(3) The invention will now be illustrated by the following examples which shall, however, not be construed as limiting the scope of the present invention.

EXAMPLES

Example 1: Double-Fluorescence-Cell Based-BoNT/A Activity ELISA

(4) Fixation of Cells

(5) 1. Remove the media/toxin solution. Add 100 l/well ice-cold methanol (20 C.) and incubate for 20 min at 20 C.

(6) Note: Perform all subsequent steps at room temperature.

(7) After Cell Fixation:

(8) 1. Remove the methanol solution and add 100 l/well PBS buffer. For longer storage (>1 day) one should add 300 l//well PBS buffer and seal the plates with parafilm. The plates should be stored in the refrigerator.

(9) 2. Remove the PBS buffer and wash the cells 3 times with 200 l/well of PBS buffer. Each step should be performed for 1 minute with gentle shaking.

(10) 3. Remove the PBS buffer and add 100 l/well of quenching buffer and incubate for 20 minutes with gentle shaking.

(11) 4. Remove the quenching buffer and wash the cells once with 300 l/well of PBS buffer for 3 minutes under gentle shaking.

(12) 5. Remove the PBS buffer, and add 200 l/well of blocking buffer and incubate for 1 hour with gentle shaking.

(13) 6. Remove the blocking buffer and add 100 l of the primary antibody mixture (antibody dilution in blocking buffer) to each well. Incubate overnight (16-18 h) with gentle shaking. The cells are simultaneously incubated with two primary antibodies: a mouse antibody specific for the BoNT/A-cleaved SNAP-25 and a polyclonal rabbit antibody that recognizes SNAP-25 (antibody for determining the total amount of SNAP 25 for normalization).
7. Remove the primary antibody mixture and wash the cells 4 times with 200 l of PBS buffer. Each step should be performed for 3 minutes with gentle shaking.
8. Remove the PBS buffer, and add 100 l of the secondary antibody mixture: HRP-conjugated anti-mouse and AP-conjugated anti-rabbit secondary antibodies (antibody dilution in blocking buffer) to each well and incubate for 2.5 to 3 hours with gentle shaking.
9. Remove the secondary antibody mixture and wash the cells 5 times with 200 l/well of PBS buffer, followed by 1 washing step with 300 l/well of HEPES buffer. Each wash step should be performed for 3 minutes with gentle shaking.
10. Remove the HEPES buffer from the plate and add 75 l of a fluorogenic substrate for horseradish-peroxidase (HRP substrate) to each well. Incubate for 50 minutes with gentle shaking. Protect the plates from direct light.
11. Add 75 l of a fluorogenic substrate for alkaline phosphatase (AP substrate) to each well and incubate for an additional 50 minutes at with gentle shaking. Protect the plates from direct light.
12. Read the plates using a fluorescence plate reader:
excitation at 540 nm; emission at 600 nm.
excitation at 360 nm; emission at 450 nm.
13. Calculation

(14) For normalization, the RFU value for cleaved SNAP-25 (fluorescence at 600 nm) is normalized to RFU of total SNAP-25 (450 nm) in each well. For better illustration of RFUs in a diagram all values are multiplied with a factor 1000 using the following equation:

(15) $\frac{\mathrm{RFU}\left(600\mathrm{nm}\right)}{\mathrm{RFU}\left(450\mathrm{nm}\right)}1000$

(16) Subsequently the resulting RFU values are averaged for each standard or sample.

(17) Reagent Preparation

(18) PBS buffer (10 mM):

(19) Phosphate buffered saline (Sigma, # P5368) (pH 7.4)

(20) Quenching Buffer:

(21) 0.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

(22) Blocking Buffer:

(23) 2% BSA in 10 mM PBS buffer (pH 7.4)+0.05% Triton X-100

(24) HEPES buffer:

(25) 50 mM HEPES (pH 7.4)

(26) HRP Substrate:

(27) 50 mM HEPES (pH 7.4)

(28) 0.007% H2O2

(29) 150 pM Amplex UltraRed

(30) AP Substrate:

(31) 25 mM Diethanolamine (pH 9.8)

(32) 2 mM MgCl.sub.2

(33) 100 l M DiFMUP

Example 2: Enhancement of Specific Uptake of Clostridial Neurotoxin Polypeptides into Cells

(34) a) iCell neurons were thawed and plated according to the Cellular Dynamics International (CDI) user manual on 96 well plates from 4 different cell batches. 24 hours (h) after plating the medium was replaced by fresh maintenance medium as described in the user manual.

(35) After further 72 h incubation time, the medium was removed and replaced by fresh medium containing BoNT/A in varying concentrations and K.sup.+-ions in a total concentration of 30 mM, (i.e. 25 mM additional K.sup.+ compared to the medium as such). After 2 hours the high-K.sup.+-medium was removed and fresh medium containing BoNT/A in varying concentrations was added to the cells.

(36) Another 70 h later, the medium was aspirated, the cells were fixed and an ELISA readout was performed as described in Example 1. The results of this protocol are given in FIG. 1, squares.

(37) b) iCell neurons were thawed and plated according to the Cellular Dynamics International (CDI) user manual on 96 well plates from 4 different cell batches. 24 hours (h) after plating the medium was replaced by fresh maintenance medium as described in the user manual.

(38) After further 72 h incubation time, the medium was removed and replaced by fresh medium containing BoNT/A in varying concentrations. After 8 hours the BoNT/A-containing-medium was removed and fresh medium without BoNT/A was added to the cells.

(39) Another 64 h later, the medium was aspirated, the cells were fixed and an ELISA readout was performed as described in Example 1. The results of this protocol are given in FIG. 1, triangles.

(40) c) iCell neurons were thawed and plated according to the Cellular Dynamics International (CDI) user manual on 96 well plates from 4 different cell batches. 24 hours (h) after plating the medium was replaced by fresh maintenance medium as described in the user manual.

(41) After further 72 h incubation time, the medium was removed and replaced by fresh medium containing BoNT/A in varying concentrations. The cells were put on a plate shaker in the incubator and were shaken at an average flow rate of 300 cm/min during toxin exposition time.

(42) Another 72 h later, the medium was aspirated, the cells were fixed and an ELISA readout was performed as described in Example 1. The results of this protocol are given in FIG. 1, circles.

(43) d) iCell neurons were thawed and plated according to the Cellular Dynamics International (CDI) user manual on 96 well plates from 4 different cell batches. 24 hours (h) after plating the medium was replaced by fresh maintenance medium as described in the user manual.

(44) After further 72 h incubation time, the medium was removed and replaced by fresh medium containing BoNT/A in varying concentrations.

(45) Another 72 h later, the medium was aspirated, the cells were fixed and an ELISA readout was performed as described in Example 1. The results of this protocol are given in FIG. 1.

CONCLUSION

(46) In sum, K.sup.+-mediated depolarization of the cells, a reduced neurotoxin polypeptide exposition time or agitation of the cells during neurotoxin polypeptide exposition facilitate comparable stability indicating kinetics of the cell based assay of Example 1 when compared to the mouse LD.sub.50 bioassay.